CN109270258A - A kind of saikosaponin D Test paper preparation method - Google Patents
A kind of saikosaponin D Test paper preparation method Download PDFInfo
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- CN109270258A CN109270258A CN201811290569.0A CN201811290569A CN109270258A CN 109270258 A CN109270258 A CN 109270258A CN 201811290569 A CN201811290569 A CN 201811290569A CN 109270258 A CN109270258 A CN 109270258A
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- saikosaponin
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N2021/7756—Sensor type
- G01N2021/7759—Dipstick; Test strip
Abstract
The invention belongs to medical monitoring device technical field more particularly to a kind of saikosaponin D Test paper preparation methods.Saikosaponin D Test paper preparation method of the present invention, a kind of test paper that can effectively detect saikosaponin D can be prepared, rapidly and accurately the presence of saikosaponin D and its content can be shown, there is the detection means for being provided with effect on the way such as examination in radix bupleuri Quality Detection and radix bupleuri, Test paper preparation is convenient, testing result is accurate, especially compounds content type is more in the combination drugs such as Chinese medicine, the influence of other compounds can be effectively avoided in the case that similar structural material content is big, the existence of correct reflection saikosaponin D, with good market application prospect.
Description
Technical field
The invention belongs to medical monitoring device technical field more particularly to a kind of saikosaponin D Test paper preparation methods.
Background technique
Saikosaponin D refers to effective effective component of bupleurum Chinese, radix bupleuri as drug important in the medicines such as Chinese medicine system it
One, it is demand product important in the market, with the medical continuous increase out of demand, people start large scale cultivating plantation radix bupleuri
To meet the market demand, but radix bupleuri qualities numerous in the market is irregular.And it is single by the conventional judgement such as appearance and smell
Method can not effectively screen the medicinal quality of reality of radix bupleuri, and the radix bupleuri product for causing buying to obtain can not achieve expected treatment
Effect, it is therefore necessary to radix bupleuri quality be analyzed, a kind of method that can effectively detect the medicinal quality of radix bupleuri is provided.
Summary of the invention
The purpose of the invention is that saikosaponin D can effectively be detected, be the practical drug effect quality of radix bupleuri by providing one kind
The detection method of reference standard is provided, this method has made a kind of saikosaponin D Test paper, being capable of convenient effective detection bavin
The presence of Hu effective component provides approach for the resolution and quality analysis of radix bupleuri.
To achieve the above object, a kind of saikosaponin D Test paper preparation method of the invention, includes the following steps:
Step 1, glass container of the preparation through overpickling or surface passivating treatment are as experiment appliance；Take 120 unit bodies
Long-pending tri-distilled water is heated to boiling, and sequentially adds 1% sodium citrate of 0.5 unit volume, 4% chlorauric acid solution and 5 unit volume
Solution, continuous heating mixed solution is until solution becomes transparent claret and no longer kept for temperature 10 minutes after variation, cooling
It is packed into glass container afterwards and prepares colloidal gold solution；
Step 2, the solution of potassium carbonate that 0.1mol/L is added in the above-mentioned colloidal gold solution of 10 unit volumes adjust pH value
To best pH value, it is slowly added to the saikosaponin D monoclonal antibody of lowest bid metering, is added after shaking 30 minutes at room temperature
10% bovine serum albumin solution is 1% up to the concentration of bovine serum albumin(BSA), and it is quiet at room temperature after twenty minutes to continue shaking
It sets 10 minutes, 10% polyglycol solution is added until Polyethylene glycol is stored at room temperature 25 minutes after being 1%；
Step 3, at 4 DEG C, 20 minutes removal deposits, use will be centrifuged with 2000r/ minutes revolving speeds under above-mentioned solution
Removal supernatant is added colloidal gold suspension and gold labeling antibody solution is made after revolving speed is centrifuged 25 minutes within 10000r/ minutes；
Step 4 will test on bonding pad after the dilution of gold labeling antibody solution, determine antibody release rate highest, route
Clear and glitch-free extension rate；Different T/C line concentration is carried out on the basis of the gold labeling antibody solution of the extension rate
Test selects colour developing to clean glitch-free T/C line concentration as benchmark；
Nitrocellulose filter is attached on bottom plate by step 5, and nitrocellulose filter lower end and bottom plate water suction end spacing be not small
In 1.5 centimetres, bonding pad, the overlapping of bonding pad lower end and nitrocellulose filter are inserted between nitrocellulose filter upper end and bottom plate
Length is 0.1 centimetre, is inserted into sample pad between bonding pad upper end and floor, and the overlapping length of sample pad lower end and bonding pad is
2mm。
Specifically, in step 1, pickling, which refers to, configures glass container by potassium bichromate, distilled water, the concentrated sulfuric acid
Thoroughly cleaned after enough time after impregnating in pickling solution, the configuration method of pickling solution be 40 unit volumes distilled water with
And 360 unit volume concentrated sulfuric acid mixed liquor in be added 7.5 unit volumes potassium bichromate be uniformly mixed；Surface passivating treatment
Method is clean with distilled water flushing after impregnating enough time in the chloroformic solution for be put into glass container 5% dichloride methane.
Specifically, in step 2, best pH value refers to, bovine serum albumin(BSA) is added in 1 unit volume of mixed solution
It solution left standstill 5 minutes, adds solution colour after 0.1 unit volume, 10% sodium chloride solution stands 2h and persistently keeps red and carbon
PH value when sour potassium additional amount is minimum；Wherein optimum PH value range is in 7-11；
Minimum meter amount refers to, saikosaponin D monoclonal is added in the mixed solution of the best pH value of 0.2 unit volume
Antibody is uniformly mixed, and solution colour persistently keeps red and radix bupleuri after adding the 10% sodium chloride standing 2h of 0.2 unit volume
Scalar when saponin D monoclonal antibody additional amount is minimum, wherein lowest bid measures range is in 0.2-0.3mg/ unit volume.
The beneficial effect is that:
Saikosaponin D Test paper preparation method of the present invention, one kind, which can be prepared, can effectively detect saikosaponin D
Test paper, rapidly and accurately the presence of saikosaponin D and its content can be shown, in radix bupleuri Quality Detection and
There is the detection means for being provided with effect on the way such as examination in radix bupleuri, Test paper preparation is convenient, and testing result is accurate, especially
Other can be effectively avoided in the case that the more similar structural material contents of compounds content type are big in the combination drugs such as Chinese medicine
The influence of compound, the correct existence for reflecting saikosaponin D, has good market application prospect.
Specific embodiment
A kind of saikosaponin D Test paper preparation method of the invention, basic step include:
Step 1, glass container of the preparation through overpickling or surface passivating treatment are as experiment appliance；Take 120 unit bodies
Long-pending tri-distilled water is heated to boiling, and sequentially adds 1% sodium citrate of 0.5 unit volume, 4% chlorauric acid solution and 5 unit volume
Solution, continuous heating mixed solution is until solution becomes transparent claret and no longer kept for temperature 10 minutes after variation, cooling
It is packed into glass container afterwards and prepares colloidal gold solution；
Step 2, the solution of potassium carbonate that 0.1mol/L is added in the above-mentioned colloidal gold solution of 10 unit volumes adjust pH value
To best pH value, it is slowly added to the saikosaponin D monoclonal antibody of lowest bid metering, is added after shaking 30 minutes at room temperature
10% bovine serum albumin solution is 1% up to the concentration of bovine serum albumin(BSA), and it is quiet at room temperature after twenty minutes to continue shaking
It sets 10 minutes, 10% polyglycol solution is added until Polyethylene glycol is stored at room temperature 25 minutes after being 1%；
Step 3, at 4 DEG C, 20 minutes removal deposits, use will be centrifuged with 2000r/ minutes revolving speeds under above-mentioned solution
Removal supernatant is added colloidal gold suspension and gold labeling antibody solution is made after revolving speed is centrifuged 25 minutes within 10000r/ minutes；
Step 4 will test on bonding pad after the dilution of gold labeling antibody solution, determine antibody release rate highest, route
Clear and glitch-free extension rate；Different T/C line concentration is carried out on the basis of the gold labeling antibody solution of the extension rate
Test selects colour developing to clean glitch-free T/C line concentration as benchmark；
Nitrocellulose filter is attached on bottom plate by step 5, and nitrocellulose filter lower end and bottom plate water suction end spacing be not small
In 1.5 centimetres, bonding pad, the overlapping of bonding pad lower end and nitrocellulose filter are inserted between nitrocellulose filter upper end and bottom plate
Length is 0.1 centimetre, is inserted into sample pad between bonding pad upper end and floor, and the overlapping length of sample pad lower end and bonding pad is
2mm。
Wherein, in step 1, pickling refers to glass container in the pickling configured by potassium bichromate, distilled water, the concentrated sulfuric acid
Thoroughly cleaned after enough time after impregnating in solution, the configuration method of pickling solution be 40 unit volumes distilled water and
The potassium bichromate that 7.5 unit volumes are added in the concentrated sulfuric acid mixed liquor of 360 unit volumes is uniformly mixed；The side of surface passivating treatment
Method is clean with distilled water flushing after impregnating enough time in the chloroformic solution for be put into glass container 5% dichloride methane；
In step 2, bovine serum albumin solution is added in 1 unit volume of mixed solution and stands 5 minutes, add
0.1 unit volume, 10% sodium chloride solution stand 2h, then solution colour persistently keeps red and potassium carbonate additional amount it is minimum when
PH value is exactly best pH value, and optimum PH value range is in 7-11；It is added in the mixed solution of the best pH value of 0.2 unit volume
Saikosaponin D monoclonal antibody is uniformly mixed, and 10% sodium chloride for adding 0.2 unit volume stands 2h, then solution colour is held
Scalar when red and minimum saikosaponin D monoclonal antibody additional amount are held in continuation of insurance is exactly lowest bid metering, and lowest bid measures model
It is trapped among 0.2-0.3mg/ml.
The specific test that saikosaponin D analogue is carried out to above-mentioned test paper, utilizes saikoside B1, saikoside
The analogue of the saikosaponin Ds such as B2, saikoside A compares detection, the unified table of its test paper after test paper detects
Now for feminine gender, the medical compound for continuing to use the structures such as scutellaria glycosides, Puerarin dissmilarity is detected, and test paper is not also negative
Property, after further carrying out specific test using plurality of Chinese and compound, saikosaponin D is only contained in bupleurum particles
In the case of, test paper is shown as positive, has good specificity.
Finally it should be noted that above embodiments are only to illustrate the technical solution of the invention, rather than to this hair
It is bright create protection scope limitation, although being explained in detail referring to preferred embodiment to the invention, this field it is general
Lead to it will be appreciated by the skilled person that can be modified or replaced equivalently to the technical solution of the invention, without departing from this
The spirit and scope of innovation and creation technical solution.
Claims (3)
1. a kind of saikosaponin D Test paper preparation method, which comprises the steps of:
Step 1, glass container of the preparation through overpickling or surface passivating treatment are as experiment appliance；Take 120 unit volumes
Tri-distilled water is heated to boiling, and sequentially adds 0.5 unit volume, 4% chlorauric acid solution and 5 unit volume, 1% sodium citrate is molten
Liquid, continuous heating mixed solution is until solution becomes transparent claret and no longer kept for temperature 10 minutes after variation, after cooling
It is packed into glass container and prepares colloidal gold solution；
The solution of potassium carbonate adjustment pH value of 0.1mol/L is added to most in step 2 in the above-mentioned colloidal gold solution of 10 unit volumes
Good pH value is slowly added to the saikosaponin D monoclonal antibody of lowest bid metering, is added 10% after shaking 30 minutes at room temperature
Bovine serum albumin solution is 1% up to the concentration of bovine serum albumin(BSA), continues shaking and stands 10 points at room temperature after twenty minutes
10% polyglycol solution is added until Polyethylene glycol is stored at room temperature 25 minutes after being 1% in clock；
Step 3, at 4 DEG C, 20 minutes removal deposits, use will be centrifuged with 2000r/ minutes revolving speeds under above-mentioned solution
Removal supernatant is added colloidal gold suspension and gold labeling antibody solution is made after revolving speed is centrifuged 25 minutes within 10000r/ minutes；
Step 4 will test on bonding pad after the dilution of gold labeling antibody solution, determine antibody release rate highest, line clear
And glitch-free extension rate；Different T/C line concentration is tested on the basis of the gold labeling antibody solution of the extension rate,
Colour developing is selected to clean glitch-free T/C line concentration as benchmark；
Nitrocellulose filter is attached on bottom plate by step 5, and nitrocellulose filter lower end is not less than with bottom plate water suction end spacing
1.5 centimetres, it is inserted into bonding pad between nitrocellulose filter upper end and bottom plate, the overlapping of bonding pad lower end and nitrocellulose filter is long
Degree is 0.1 centimetre, is inserted into sample pad between bonding pad upper end and floor, and the overlapping length of sample pad lower end and bonding pad is
2mm。
2. a kind of saikosaponin D Test paper preparation method according to claim 1, which is characterized in that in step 1, acid
Wash refer to by glass container by potassium bichromate, distilled water, concentrated sulfuric acid configuration pickling solution in impregnate after after enough time it is thorough
Bottom cleaning, the configuration method of pickling solution is the concentrated sulfuric acid mixed liquor of the distilled water and 360 unit volumes in 40 unit volumes
The middle potassium bichromate that 7.5 unit volumes are added is uniformly mixed；The method of surface passivating treatment is that glass container is put into 5% dichloro
Change clean with distilled water flushing after impregnating enough time in the chloroformic solution of methane.
3. a kind of saikosaponin D Test paper preparation method according to claim 1, which is characterized in that in step 2, most
Good pH value refers to, bovine serum albumin solution is added in 1 unit volume of mixed solution and stands 5 minutes, adds 0.1 unit bodies
Product 10% sodium chloride solution stand 2h after solution colour persistently keeps red and potassium carbonate additional amount it is minimum when pH value；Wherein most
Good pH range is in 7-11；
Minimum meter amount refers to, saikosaponin D monoclonal antibody is added in the mixed solution of the best pH value of 0.2 unit volume
It is uniformly mixed, solution colour persistently keeps red and saikosaponin D after adding the 10% sodium chloride standing 2h of 0.2 unit volume
Scalar when monoclonal antibody additional amount is minimum, wherein lowest bid measures range is in 0.2-0.3mg/ml.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811290569.0A CN109270258A (en) | 2018-10-31 | 2018-10-31 | A kind of saikosaponin D Test paper preparation method |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201811290569.0A CN109270258A (en) | 2018-10-31 | 2018-10-31 | A kind of saikosaponin D Test paper preparation method |
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| CN109270258A true CN109270258A (en) | 2019-01-25 |
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Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101458254A (en) * | 2008-12-03 | 2009-06-17 | 周炬华 | Clenobuterol hydrochloride detecting test paper and detecting method thereof |
| CN101539578A (en) * | 2009-02-17 | 2009-09-23 | 李红玉 | Colloidal gold test strip for testing melamine content |
| CN101487843A (en) * | 2009-03-06 | 2009-07-22 | 关一夫 | Fast urine HIV detection diagnosis test paper and method for producing the same |
| CN104267109A (en) * | 2014-07-31 | 2015-01-07 | 甘肃中天药业有限责任公司 | Radix bupleuri medicinal material detection method |
| CN104459118A (en) * | 2014-12-31 | 2015-03-25 | 浙江理工大学 | Method for preparing double antibody sandwich method test paper for antique silk fabric |
| CN106568965A (en) * | 2016-10-14 | 2017-04-19 | 广州安诺食品科学技术有限公司 | Auramine O colloidal gold test card and preparation method thereof |
| CN108196054A (en) * | 2017-07-27 | 2018-06-22 | 数字本草中医药检测有限公司 | A kind of test strips for detecting glycyrrhizic acid and its preparation method and application |
Non-Patent Citations (4)
| Title |
|---|
| MORINAGA,O等: "Visual detection of saikosaponins by on-membrane immunoassay and estimation of traditional Chinese medicines containing Bupleuri radix", 《BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS》 * |
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